Site-specific immobilization of biomolecules by a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole

Article abstract of DOI:10.1039/c3cc43566k

We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.

Full text of DOI:10.1039/c3cc43566k

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Site-specific immobilization of biomolecules by a
biocompatible reaction between terminal cysteine and
2-cyanobenzothiazole†
Cite this: Chem. Commun., 2013,
49, 8644
Received 13th May 2013,
Accepted 29th July 2013
a
b
Ping Wang,z Chong-Jing Zhang,z Ganchao Chen,a Zhenkun Na,b Shao Q. Yaob
and Hongyan Sun*ac
DOI: 10.1039/c3cc43566k
www.rsc.org/chemcomm
We report herein a new site-specific microarray immobilization
Despite the aforementioned advancement, there is still
method based on a biocompatible reaction between terminal cysteine growing interest in the development of novel immobilization
and 2-cyanobenzothiazole (CBT). This immobilization strategy has methods for continuous expansion of microarray applications.
been successfully applied to anchor small molecules, peptides and An ideal immobilization method should fulfil a number of criteria.
proteins onto microarrays.
First, the conditions for immobilization should be mild, e.g. room
temperature, physiological pH and without organic co-solvents.
Microarray technology has transformed biomedical research in Biomolecules such as proteins are rather fragile, and both their
recent years.1 Over the last two decades, it has become a robust tertiary structure and biological functions may be perturbed under
and powerful screening platform for a wide range of biological harsh immobilization conditions. Secondly, the immobilization
applications, including studies of molecular interactions, drug method should be highly selective. Given that multiple functional
discovery and disease diagnosis.2 The compound immobilization groups are present in peptides, small molecules and proteins, a
strategy employed is crucial for successful implementation of micro- non-selective method could result in inhomogeneous immobiliza-
array screening. Most common immobilization strategies could be tion, leading to multiple orientations of the biomolecules.
grouped into those based on non-covalent immobilization, random Consequently, the binding site of the biomolecules may be blocked
covalent immobilization or site-specific covalent immobilization. and their molecular recognition can be disrupted. Finally, the
Among them, site-specific covalent immobilization approaches have immobilization method should be efficient. Since the immobiliza-
captured much attention because they ensure uniform display and tion process is performed on the solid surface, where reaction
optimum orientation of biomolecules upon immobilization, thereby kinetics is usually lower than that in solution, only a highly efficient
facilitating subsequent interactions of these biomolecules and their method would ensure maximum compound immobilization.
analytes during screening.
With these criteria in mind, we sought to develop a new
Over the years, a number of chemoselective reactions, approach for general immobilization under mild conditions. In
such as Diels–Alder reaction,3a native chemical ligation,3b click 2009, Rao’s group reported an efficient biocompatible reaction
chemistry,3c Staudinger ligation3d,e and thiol–ene chemistry,3f have between a terminal cysteine and 2-cyanobenzothiazole (CBT).5
been applied for site-specific immobilization of biomolecules The group demonstrated that the reaction could proceed in PBS buffer
in a microarray. A recent strategy developed by Waldmann’s without the use of any catalyst. It was reported that the overall rate of
group also involved the use of oxime ligation, in which oxyamine- this reaction was much faster than that of the well-established copper-
functionalized proteins were successfully anchored onto ketone- free click reaction.5 The reaction has been successfully employed in
derivatized glass slides chemoselectively under physiological selective labeling of terminal cysteine-containing proteins on cell
conditions (pH = 7.0).4
membranes. This useful reaction was later extended to several other
applications, such as controlled assembly of nanostructures in live cells,
site-specific incorporation of 18F for PET imaging, etc.6 In this study, we
report, for the first time, that this reaction could serve as a versatile and
robust immobilization approach for site-specific attachment of different
biomolecules on microarrays (Scheme 1). We want to point out that,
although copper(I) catalyzed click chemistry (CuAAC) has been widely
utilized in microarray immobilization,1,3c the usage of the copper(I)
catalyst can lead to the damage of biomolecules.7 While in our method,
the immobilization conditions are very mild without using any catalyst,
which eliminate the possibility of damaging biomolecules.
a Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee
Avenue, Kowloon, Hong Kong, P. R. China. E-mail: hongysun@cityu.edu.hk;
Fax: +852 3442 0522; Tel: +852 3442 9537
b Department of Chemistry, National University of Singapore, 3 Science Drive 3,
Singapore 117543
c Key Laboratory of Biochip Technology, Biotech and Health Centre,
Shenzhen Research Institute of City University of Hong Kong, Shenzhen,
P. R. China 518057
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc43566k
‡ These authors contributed equally to this work.
c
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Fig. 1 Various concentrations of Cys–TER were applied to CBT-functionalized
slides (0.2, 0.5, 2, 20, 200 and 2000 mM). (a) Fluorescence microarray images. (b)
The structure of Cys–TER used in this study. (c) The plot of relative fluorescence
intensity of (a).
Scheme 1 Schematic illustration of the immobilization of terminal cysteine-
containing biomolecules onto 2-cyanobenzothiazole (CBT)-functionalized slides
in this study. Su = succinimide.
with increasing concentrations of Cys–TER. Further data analysis
revealed that the surface of CBT-coated slides started to become
saturated at a concentration of around 20 mM. When the concen-
tration was above 200 mM, the slide surface became completely
saturated. To rule out the possibility of non-specific binding,
a negative control, amine-TER was included in our experi-
ments. No noticeable fluorescence signal for amine-TER was
observed under the same experimental conditions (see ESI,†
Fig. S2), indicating that this newly developed microarray immo-
bilization method was highly selective.
Scheme 2 Synthetic scheme of a glycine–CBT molecule. (a) KNO3ꢀH2SO4, ice,
1 h, 78.8%; (b) SnCl2, ethanol, HCl, reflux, 57.9%; (c) KCN, DMSO, 24.9%; (d)
isobutyl chlorformate, Boc-Gly, NMM, 57.8%; (e) TFA–DCM, 22.6%.
We next studied the immobilization of more delicate bio-
molecules, i.e. proteins. We used EGFP as our model protein in
the study. EGFP with an N-terminal cysteine was expressed and
In our setup, synthesis of glycine–CBT (6) was first carried out purified by reported procedures (Fig. 2a).9 Specifically, EGFP was
based on the reported method.8 As depicted in Scheme 2, nitration of cloned into the pTwin1 vector to generate pTwin1-intein-EGFP,
2-chlorobenzothiazole, 1, was performed under acidic conditions. The which contains an intein fused to the N-terminal residue of the
resulting nitro derivative, 2, was then reduced to its amine form, 3, EGFP. An extra N-terminal cysteine residue was introduced into
using SnCl2 (57.9% yield). The installation of a cyanide group was EGFP through the PCR method. pH-induced intein cleavage resulted
achieved by reacting 3 with KCN at high temperature. Subsequently, a in the release of the EGFP containing a cysteine residue at its
Boc-glycine linker was introduced at the amine group of 4 using N-terminus. Coomassie staining analysis revealed that the protein
isobutyl chloroformate as the activating reagent. Finally, the Boc was sufficiently pure after purification (Fig. 2b and Fig. S5, ESI†).
group was removed with TFA to yield the target molecule, 6. The Various concentrations of EGFP were then immobilized on CBT-
structural identification of glycine–CBT 6 was confirmed by 1H NMR, functionalized slides in a humid chamber. Microarray images clearly
13C NMR and ESI-MS respectively (ESI†).
In the next step, we moved forward to produce CBT-functionalized
slides. N-Hydroxysuccinimide (NHS) ester-functionalized slides
were first fabricated following the previously established proto-
col (ESI†). A solution of glycine–CBT in DMF was subsequently
applied to NHS-coated slides under basic conditions to produce
CBT-functionalized slides.
To establish the method, we first tested the immobilization
efficiency of this strategy using small molecule dyes (Fig. 1). A
cysteine-containing dye, cysteine–tetraethylrhodamine (Cys–TER)
(Fig. 1b), was synthesized and reacted with glycine–CBT in PBS buffer
(pH = 7.4). LC-MS analysis indicated that the reaction occurred rapidly
and was complete within 1 hour (see ESI,† Fig. S1). No side reaction
was observed. Encouraged by this result, we carried out small
molecule immobilization in our preliminary experiments. Different
concentrations of Cys–TER ranging from 0.2 to 2000 mM were spotted
in duplicate onto CBT-functionalized slides, followed by incubation in
a humid chamber for 3 hours. Excess dyes were then washed away,
Fig. 2 Immobilization of EGFP onto a CBT-functionalized slide. (a) Preparation of
N-terminal cysteine-containing EGFP via the intein-mediated cleavage method. (b) SDS-
PAGE analysis of N-His-EGFP and N-Cys-EGFP. (c) Different concentrations of N-Cys-EGFP
and the slides were imaged using a microarray fluorescence scanner.
As shown in Fig. 1a, the fluorescence intensity of the spots increased
and N-His-EGFP were immobilized onto CBT-functionalized slides. (d) Time-dependent
immobilization of N-terminal cysteine-containing EGFP onto microarrays.
c
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non-cysteine-containing RGD peptides display a similar growth
pattern to CBT-functionalized slides (Fig. 3b and d), proving that
there is no immobilization of this peptide. The method described
herein thus proves that cysteine-containing peptide substrates can
be covalently linked to CBT-coated slides and used for cell adhesion
studies. It can provide a facile approach to screen peptide ligands
capable of promoting cellular adhesion and growth.
In summary, we have developed a new method for site-
specific microarray immobilization of biomolecules, including
small molecules, peptides and proteins. The reaction can take
place under very mild conditions, and it is highly selective
towards terminal cysteine-containing molecules. We believe
that this new method will be a useful tool to current and future
microarray applications.
The authors would like to thank for the financial support
from National Natural Science Foundation of China (Project
Fig. 3 Cell adhesion study of slides immobilized with different peptide sub-
strates. Microscopic images of cells grown on slides (a) coated with CRGD
peptides, (b) coated with RGD peptides, (c) coated with CGGG peptides, (d) No. 21202137) and the City University of Hong Kong SRG grant
CBT-functionalized slides.
(7002865 and 7002721).
Notes and references
indicated that the fluorescence intensity increased with increasing
concentrations of the protein (Fig. 2c). In order to prove that the
reaction was site-specific toward only the N-terminal cysteine in the
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8646 Chem. Commun., 2013, 49, 8644--8646
This journal is The Royal Society of Chemistry 2013